A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

Targeted protein degradation promises to enable small molecule-mediated modulation of currently undrugged proteins. While the well-characterized E3 ligases CRBN and VHL have successfully promoted the degradation of many proteins of interest, there are approximately 600 additional E3 ligase family members that may offer improved activity, substrate selectivity, or tissue distribution; however, characterizing the ability of these many ligases to promote targeted protein degradation has proven challenging. Here, we report the development of a rapid method to evaluate the ability of recombinant E3 ligase components to support the degradation of neo-substrates. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method using covalent E3-target binder complexes of VHL-JQ1 and VHL-dasatinib and show the degradation of Brd4 and Lyn kinase, respectively. Applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase not previously shown to degrade neo-substrates, we demonstrated the ability of this method to rapidly validate an uncharacterized ligase for degradation of neo-substrates

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of gamma-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

International audiencePropofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured approximately 4% of the synaptosomal proteome, including the unbiased capture of five alpha or beta GABAA receptor subunits. Lack of gamma2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for alpha/beta than gamma-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and alpha/beta cavity residues but not gamma cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity

A Strategy to Assess the Cellular Activity of E3 Ligase Components against Neo-Substrates using Electrophilic Probes

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While well-characterized E3 ligases such as CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution. Characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple maleimide-thiol chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or tyrosine kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F-box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only ligase receptors but also adaptors can be directly hi-jacked for neo-substrate degradation

Role for the Propofol Hydroxyl in Anesthetic Protein Target Molecular Recognition

Propofol is a widely used intravenous general anesthetic. We synthesized 2-fluoro-1,3-diisopropylbenzene, a compound that we call “fropofol”, to directly assess the significance of the propofol 1-hydroxyl for pharmacologically relevant molecular recognition in vitro and for anesthetic efficacy in vivo. Compared to propofol, fropofol had a similar molecular volume and only a small increase in hydrophobicity. Isothermal titration calorimetry and competition assays revealed that fropofol had higher affinity for a protein site governed largely by van der Waals interactions. Within another protein model containing hydrogen bond interactions, propofol demonstrated higher affinity. In vivo, fropofol demonstrated no anesthetic efficacy, but at high concentrations produced excitatory activity in tadpoles and mice; fropofol also antagonized propofol-induced hypnosis. In a propofol protein target that contributes to hypnosis, α1β2γ2L GABA_A receptors, fropofol demonstrated no significant effect alone or on propofol positive allosteric modulation of the ion channel, suggesting an additional requirement for the 1-hydroxyl within synaptic GABA_A receptor site(s). However, fropofol caused similar adverse cardiovascular effects as propofol by a dose-dependent depression of myocardial contractility. Our results directly implicate the propofol 1-hydroxyl as contributing to molecular recognition within protein targets leading to hypnosis, but not necessarily within protein targets leading to side effects of the drug